We propose to investigate the observed positional effect of mating type gene in the yeast Saccharomyces. There are three loci in Saccharomyces consisting of one of two possible controlling elements that can determine cell type. At one of these loci, MAT, this information is expressed to give rise to the mating type of the cell. At the other two loci, HML and HMR, this same information is phenotypically and transcriptionally silent. This is true even though the sequences at MAT, HMR, and HML can be identical well in excess of the coding regions contained within the element. Transcriptional repression of HML, and HMR requires the transactive gene products of four unlinked loci, SIR1 through SIR4. In addition, repression of HML or HMR requires the integrity of two sites (designate "e" and "i") at each locus, which in both cases flank that region of DNA common to the three mating type loci. Since the "e'" and "i" sites lie considerable distances from the promoters of the genes they regulate, this mode of regulation obviously involves action at a distance. Current information suggests that repression is probably mediated by alterations in chromatin structure, changes in local superhelical density of the chromsome, or both. We propose to analyze the mechanism of transcriptional repression at HML by (1) additional in vitro mutagenesis of the "e" site adjacent to HML; (2) isolation and characterization of the SIR1 through SIR4 gene products; (3) examination of the chromatin spanning HML during transition from an active to an inactive state; and (4) determining the temporal order of action of the SIR gene products. The results from these experiments should provide significant insights into the role of chromatin structure in controlling expression of eucaryotic cells. In addition these results can provide a model system for evaluating positional effects observed in higher cells as well as the role of alterations in chromatin structure as a function of normal and abnormal development.